JPS6332767B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention comprises about 2 to 5 parts by weight of partially hydrolyzed gelatin with an average molecular weight of about 3000, about 2 to 55 parts by weight of hexitol, about 0.5 to 1.7 parts by weight of a virus culture medium, and a pH adjusted to about 6.0 to 6.5. An inactivated or attenuated virus vaccine stabilizer consisting essentially of an amount of a physiologically acceptable buffer effective to Examples of live viruses are measles, mumps or rubella, chickenpox, polio or hepatitis, or a combination of two or more such viruses. Hydrolyzed gelatin is used to provide a soluble, non-gelling proteinaceous matrix with little or no pyrogenicity or antigenicity. Partially hydrolyzed gelatin refers to gelatin that has been partially hydrolyzed to yield partially hydrolyzed gelatin having an average molecular weight of about 3000. This gelatin hydrolysis product has almost the same amino acid composition as gelatin. Unlike gelatin, which forms a gel but is insoluble in cold water, hydrolyzed gelatin does not form a gel but dissolves in cold water and other common liquids such as milk and orange juice. Contains hydrolyzed gelatin to a concentration of approximately 10%. Above about 10% concentration, the viscosity increases gradually. At about 50% concentration the solution is quite sticky. The typical amino acid composition of hydrolyzed gelatin is as follows: Alanine 8.5% Arginine 7.9% Aspartic acid 5.7% Cystine 0.08% Glutamic acid 9.5% Glycine 22.8% Histidine 0.77% Hydroxyproline 13-14% Isoleucine 1.3% Leucine 2.9% Lysine 4.2% Methionine 0.78% Phenylalanine 2.0% Proline 13.8% Serine 3.3% Threonine 1.9% Tyrosine 0.40% Valine 2.4% Partially hydrolyzed gelatin contains proteolytic enzymes such as papain, chymopapain and bromelin. by enzymatic hydrolysis of gelatin, but other known hydrolysis methods such as acid hydrolysis can be used. A suitable hydrolyzed gelatin is obtained from Wilson & Company, Inc., Calmette City, IL under the trade name SOL-U-PRO. Polyhydric alcohols having 6 carbon atoms are, for example, sorbitol, mannitol or dulcitol. Sorbitol is preferred. The acidic buffer can be any physiologically acceptable buffer that maintains the desired pH of about 6 to 6.5, such as phosphate buffer, acetate buffer or citrate buffer. Phosphate buffers are preferred. The stabilizer is diluted with about 3 to 8 times its weight, preferably about 5.5 times its weight, of distilled water before use. A cell culture medium is a nutrient medium that allows cells to grow in vitro. Some specific nutrient media are e.g. Medium 199, Morgan et al., Proc.Soc.Exp.
Biol. & Med. Vol. 73, pp. 1-8, 1950; Basal Medium Eagle, Eagle;
Science Vol. 122, pp. 501-504, 1955; In Vitro, Vol. 6, No. 2, 1970
year; Dulbecco's modified Eagle medium (Dulbecco's
Modified Eagle's Medium). Grubeko et al., Virology Vol. 8, p. 396, 1959; Smith et al.
J.Virol, Vol. 12, pp. 185-196
1960; In Vitro Vol. 6, No. 2, 1970; Minmum Essential Medium (Eagle), Science, Vol. 130, No. 432 (1959)
) and RPMI medium, Mole et al., Vol. 199, No.
pp. 519-524, 1967; in Vitro (in
Vitro) Volume 6, No. 2, 1970. It has been experimentally found that the stabilizer composition of the present invention is effective in improving storage stability when it contains the following components in the following amounts: parts by weight of component partially hydrolyzed. gelatin 2 to 5 Polyhydric alcohol 2 to 55 Nutrient medium (solid) 0.5 to 1.7 Physiologically acceptable buffer to adjust pH to 6.0 to 6.5 Sufficient amount For liquid vaccines, sorbitol is generally used in amounts towards the upper end of the range. whereas in lyophilized vaccines sorbitol is present in amounts towards the lower end of the range. The specific formulation for the viral vaccine of the invention is as follows. Formulation B is suitable for lyophilized vaccines.

[Table] ni ni
Furthermore, the stabilizer optionally but preferably contains small amounts of NaHCO ₃ and phenol red.
In the case of the previous formulation, NaHCO ₃ may be present in an amount of about 1.2 g and phenol red in an amount of about 0.01 g. While specific formulations are described above, variations in the proportions and concentrations of each component are anticipated. One volume of bulk vaccine is usually diluted with about 2 to 12 volumes of stabilizer. The following examples illustrate, but do not limit, the invention. Example 1 80 ml of measles virus concentrate (virus content: titer 4.2TCID50/0.1 ml) stored at -70°C was dissolved at 25°C in a water bath and then kept at 4-8°C. The liquid viral concentrate was then divided into two aliquots of 40 ml each. (a) One 40 ml aliquot from this virus fluid was diluted into 210 ml of sterile stabilizer of Formulation B as described above. The formulation was carried out under aseptic conditions and under a laminar flow hood. 10.5 mg of neomycin was added to the specimens to prevent microbial growth. Dispense the diluted vaccine into 2 ml glass ampoules (0.7 ml vaccine per ampoule)
Immediately sealed with flame and stored at 4-8°C. (b) A second 40 ml aliquot was run as the first except that standard commercially available vaccine diluent (SPGA) was used in place of the stabilizer in Formulation B. The storage stability of the vaccine is listed in the following table:

[Table]
Example 2 32 ml of measles virus concentrate (virus content: titer 3.5TCID50/0.1 ml) stored at -70°C was dissolved at 25°C in a water bath and then kept at 4-8°C.
The liquid viral concentrate was divided into two aliquots of 16 ml each. (a) One 16 ml aliquot from this virus fluid was diluted in 48 ml of sterile stabilizer of Formulation B as described above. The formulation was carried out under aseptic conditions and under a laminar flow hood. 2.5 mg of neomycin was added to the specimens to prevent microbial growth. The diluted vaccine was dispensed into 2 ml glass ampoules (0.7 ml per ampoule), immediately flame sealed and stored at 37°C. (b) A second 16 ml aliquot was operated as the first except that the stabilizer of Formulation A was used in place of the stabilizer of Formulation B. The storage stability of the vaccine is listed in the following table:

[Table] Example 3 80 ml of measles virus concentrate (virus content: titer 4.2TCID50/0.1 ml) stored at -70°C was dissolved at 20°C in a water bath and then kept at 4-8°C. The liquid viral concentrate was divided into two portions of 40 ml each. (a) One 40 ml aliquot from this virus fluid was diluted in 210 ml of sterile stabilizer of Formulation B as described above. The formulation was carried out under aseptic conditions and under a laminar flow hood. 10.5 mg of neomycin was added to the specimens to prevent microbial growth. Dispense the diluted vaccine into 3 ml glass vials (0.7 vaccine per vial), lyophilize and stopper 37
Stored at °C. (b) A second 40 ml vial aliquot was operated like the first except that a standard commercial diluent (Medium 199 containing SPGA) was used in place of the Formulation B stabilizer.

[Table]
Example 4 The lyophilized vials produced in Example 3 were regenerated in distilled water (0.7 ml per vial) and 2
Stored at ~8°C. The storage stability of these vaccines is listed in the following table:

[Table]

Claims (1)

[Scope of Claims] 1. About 2 to 5 parts by weight of partially hydrolyzed gelatin having an average molecular weight of about 3000, about 2 to 55 parts by weight of hexitol.
an inactivated or attenuated viral vaccine stabilizer consisting essentially of parts by weight, about 0.5 to 1.7 parts by weight of virus culture medium, and an amount of a physiologically acceptable buffer effective to adjust the pH to about 6.0 to 6.5. . 2. The stabilizer according to claim 1, wherein the hexitol is sorbitol. 3. The stabilizer according to claim 1, wherein the buffer is a phosphate buffer. 4. The stabilizer of claim 1, wherein the virus is measles, mumps, rubella, chickenpox, polio or hepatitis, herpes simplex 1, herpes simplex 2, or a combination thereof. 5 Gelatin with partially hydrolyzed stabilizer
3.6 parts by weight, approximately 3.6 parts by weight of sorbitol, medium 199
The stabilizer of claim 1 consisting essentially of about 1.1 parts by weight of phosphate buffer and an amount effective to adjust the pH to about 6.0 to 6.5. 6 Gelatin with partially hydrolyzed stabilizer
3.6 parts by weight, approximately 53 parts by weight of sorbitol, approximately 199 parts by weight of medium
The stabilizer of claim 1 consisting essentially of 1.1 parts by weight of phosphate buffer and an amount effective to adjust the pH to about 6.0 to 6.5. 7. One volume of inactivated or reduced virus bulk vaccine is combined with about 2 to 5 parts by weight of partially hydrolyzed gelatin having an average molecular weight of about 3000, about 2 to 55 parts by weight of hexitol, and about 0.5 to 1.7 parts by weight of virus culture medium. Inactivation or inactivation characterized by diluting in about 2 to 12 volumes of a vaccine stabilizer consisting essentially of parts by weight and an amount of a physiologically acceptable buffer effective to adjust the pH to about 6.0 to 6.5. Method for stabilizing attenuated virus vaccines. 8 About 2 to 5 parts by weight of partially hydrolyzed gelatin with an average molecular weight of about 3000, about 2 to 55 parts by weight of hexitol
an inactivated or attenuated viral vaccine stabilizer consisting essentially of parts by weight, about 0.5 to 1.7 parts by weight of virus culture medium, and an amount of a physiologically acceptable buffer effective to adjust the pH to about 6.0 to 6.5. A method for stabilizing an inactivated or attenuated virus vaccine, either by regenerating the lyophilized vaccine with water or by freezing the vaccine containing the stabilizer and then thawing it.